HIV reverse transcriptase (RT) is the target of many key anti-AIDS drugs. Both nucleoside and nonnucleoside RT inhibitors are used as effective drugs for treating AIDS, but success can be limited by the emergence of drug-resistant viral variants. Promising non-nucleoside RT inhibitors have been developed through structure-based methods and we propose to carry out crystallographic studies that could enable structure-based improvement of additional classes of RT inhibitors. Some of the studies are designed to test recently established models for HIV-1 RT resistance to nucleoside analogs in which terminal nucleotides can be excised via a pyrophosphorolytic mechanism involving ATP as the pyrophosphate donor, releasing a dinucleoside tetraphosphate product. The use of strategically modified protein and nucleic acids will enhance the quality and incisiveness of the structural and mechanistic studies. The proposed structural studies will also enhance our understanding of inhibition and drug-resistance mechanisms. Specifically, we propose to determine the following structures: 1) wild-type and drug-resistant mutant HIV-1 RT compiexed with selective inhibitors of RNase H; 2) AZT-resistant mutants of HIV-1 RT complexed with template-primer in the "excision" position (positioned in a pre-translocation position) and bound ATP and specifically modified ATP analogs; 3) wild-type and drug-resistant mutant HIV-1 RT complexed with template-primer and dinucleoside tetraphosphate analogs as potential inhibitors; and 4) wild-type and K65R mutant HIV-1 RT complexed with template-primer and the acyclic nucleotide monophoshonate tenofovir (PMPA, Viread) both as binary (incorporated at the primer terminus) and ternary (dideoxy-terminated template-primer and the biologically active tenofovir diphosphate) forms. The proposed work relies on collaborations with Dr. Stephen Hughes (Project 2; production of wild-type and mutant RT including enzymes with strategically placed cysteine residues, nucleoside analog inhibition and resistance mechanisms); Dr. Michael Parniak (Project 3; chemical synthesis of RNase H inhibitors, mechanisms of RNase H inhibition and resistance); and Dr. Roger Jones (Project 4; synthesis of tailored nucleic acid reagents for structural and mechanistic studies, synthesis of dinucleoside tetraphosphate analogs as potential inhibitors of HIV-1 RT).